The long term objective of our work is to understand how nicotinic receptors participate in signaling in the nervous system. Nicotinic receptors are found in both the peripheral and central nervous systems, where they are important for a variety of functions, including control of movement, control of excitability, cognition, memory, anesthesia, analgesia, and reward. The specific aim of the current proposal is to explore, using a combination of experimental and modeling approaches, fundamental mechanisms by which nicotinic receptors participate in millisecond time scale signaling. These experiments will be done in a highly accessible model synapse in the embryonic chicken ciliary ganglion. Fast synapses generally operate by the release of quanta of transmitter, via exocytosis, at highly specialized sites on a nerve terminal and their interaction with high-density clusters receptors in the postsynaptic membrane opposite those sites. In the ciliary ganglion, however, there are large numbers of receptors not located opposite specialized sites from which transmitter is known to be released. The rapid time course with which these receptors are activated following release suggests that transmitter must be released directly on them;such release is therefore ectopic, or non-synaptic. A recent modeling study, based on numerical simulations performed on accurate three-dimensional representations of the synaptic volume (MCell), has supported the possibility of ectopic release in this system. In the current proposal we will refine the MCell model, using a variety of measurements on geometry and on receptor distribution, obtained using immuno electron microscopy, and will subject ectopic and conventional models to increasingly demanding tests of their ability to recapitulate actual data sets obtained using whole cell recording methods. We will also test whether transmitter release in this system has the characteristics found for another synapse that has been recently shown to operate by ectopic release. These studies should increase our understanding of the fundamental process by which nicotinic receptors on nerve cells participate in rapid time scale signaling. These studies may also be of use in understanding the basis of pathological states brought about, or exacerbated by, loss or alteration of receptor function (e.g., seizure disorders, neurodegenerative disease).